Research On The Immunoassay Methods Of Environmental Estrogen Bisphenol A

Bisphenol A (BPA) is a kind of environmental hormone substance which was found from food packaging materials in the recent years.A large number of studies have shown that BPA has a series of adverse effects on the nervous system, the immune system, reproductive system of mammals and threats to our health. Therefore, developing a rapid and convenient, accurate and sensitive detection technology of BPA has important significance to protect human health and the ecological environment. For this reason, high specific polyclonal antibodies against BPA were prepared, and three kinds of immune detection method of BPA residues were established in our research. The detection methods included indirectly competitive ELISA (ciELISA), directly competitive ELISA (cdELISA) and ciELISA marked by immunomagnctic beads. In addition, an ELISA kit for detecting BPA was also developed and used to test the BPA residues in food plastic bags, disposable plastic and paper cups.The diimine carbonization methods were used to synthesize immunogen BHPVA-OVA and coating antigen BHPVA-BSA by hapten4,4-Bis(4-hydroxyphenyl) valeric acid(BHPVA) being conjugated to ovalbumin (OVA) and bovine serum albumin (BSA), respectively. The results showed the ratios of hapten to OVA and BSA were16:1and12:1by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). After New Zealand white rabbits were immunized with BHPVA-OVA, the polyclonal antibodies against BPA were prepared, whose ELISA titer was1:1×107. A ciELISA was developed for BPA detection, which has a detection limit (IC10) of0.0414ng/mL and a linear working range of0.024-100ng/mL. The regression equation was y=9.0824In x+38.912, R2=0.9977. The Immunomagnetic beads (IMB) were prepared by xMag-magnetic beads marking PcAb against BPA, and a ciELISA marked by immunomagnctic beads was established for BPA detection, which has a detection limit (IC10) of0.006ng/mL and a linear working range of0.0001-1ng/mL. The regression equation wasy=3.6309In x+28.466, R2=0.9863. A horseradish peroxidase (HRP) labeled antibody was synthesized by the sodium periodate reaction, then a cdELISA was developed. Quantization of the BPA was linear from0.024to100ng/mL, and the hemi-inhibitory concentration (IC5o) was16.21ng/mL, while the detection limit (IC10) was0.036ng/mL.According to the ciELISA established in the study, we developed a ELISA kit for detecting BPA.We choose JET、CBS (pH9.6)coating buffer37℃/2h or4℃/12h of coating time and temperture,5%skim milk powder blocking buffer as experimental conditions.the detection limit (IC10) of the ELISA kit was0.0414ng/mL and a linear working range was0.024-100ng/mL. The regression equation was y=9.0824In x+38.912, R2=0.9977, and the hemi-inhibitory concentration (IC50) was3.39ng/mL. When50and100ng/mL BPA was added in the recovery tests, the recovery ratios of BPA were72.76%and90%in double-distilled water blank samples, respectively. The BPA residues were found in all eight kinds of food plastic bags by ELISA kit, and migration quantities of BPA ranged from0.049to0.31ng/mL with the intra-and inter-assay CV below12.48%. The experimental results indicated the established ELISA kit was effectively used to detect BPA migrations in food plastic bags, and the method had not only high sensitivity but also good stability. In addition, BPA residues were also found in disposable plastic cups and paper cups.